Publications

Zoonotic infectious diseases have been an important concern to humankind for more than 10,000 years. Today, approximately 75% of newly emerging infectious diseases (EIDs) are zoonoses that result from various anthropogenic, genetic, ecologic, socioeconomic, and climatic factors. These interrelated driving forces make it difficult to predict and to prevent zoonotic EIDs. Although significant improvements in environmental and medical surveillance, clinical diagnostic methods, and medical practices have been achieved in the recent years, zoonotic EIDs remain a major global concern, and such threats are expanding, especially in less developed regions. The current Ebola epidemic in West Africa is an extreme stark reminder of the role animal reservoirs play in public health and reinforces the urgent need for globally operationalizing a One Health approach. The complex nature of zoonotic diseases and the limited resources in developing countries are a reminder that the need for implementation of Global One Health in low-resource settings is crucial. The Veterinary Public Health and Biotechnology (VPH-Biotec) Global Consortium launched the International Congress on Pathogens at the Human-Animal Interface (ICOPHAI) in order to address important challenges and needs for capacity building. The inaugural ICOPHAI (Addis Ababa, Ethiopia, 2011) and the second congress (Porto de Galinhas, Brazil, 2013) were unique opportunities to share and discuss issues related to zoonotic infectious diseases worldwide. In addition to strong scientific reports in eight thematic areas that necessitate One Health implementation, the congress identified four key capacity-building needs: (1) development of adequate science-based risk management policies, (2) skilled-personnel capacity building, (3) accredited veterinary and public health diagnostic laboratories with a shared database, and (4) improved use of existing natural resources and implementation. The aim of this review is to highlight advances in key zoonotic disease areas and the One Health capacity needs.

Tuberculosis (TB) is the number one bacterial killer worldwide and the current increase in type 2 diabetes mellitus patients (DM), particularly in countries where TB is also endemic, has led to the re-emerging importance of DM2 as a risk factor for TB. There is an urgent need to implement strategies for TB prevention among the millions of DM patients exposed to Mycobacterium tuberculosis (Mtb) worldwide, but knowledge is limited on how and when DM2 alters the natural history of this infection. In this review we summarize the current epidemiological, clinical and immunologic studies on TB and DM and discuss the clinical and public health implications of these findings. Specifically, we evaluate the mechanisms by which DM patients have a higher risk of Mtb infection and TB development, present with signs and symptoms indicative of a more infectious TB infection, and are more likely to have adverse TB treatment outcomes, including death. Emphasis is placed on type 2 DM given its higher prevalence in contemporary times, but the underlying role of hyperglycemia and of type 1 DM is also discussed.

Macrophages, the major host cells harboring Mycobacterium tuberculosis (M.tb), are a heterogeneous cell type depending on their tissue of origin and host they are derived from. Significant discord in macrophage responses to M.tb exists due to differences in M.tb strains and the various types of macrophages used to study tuberculosis (TB). This review will summarize current concepts regarding macrophage responses to M.tb infection, while pointing out relevant differences in experimental outcomes due to the use of divergent model systems. A brief description of the lung environment is included since there is increasing evidence that the alveolar macrophage (AM) has immunoregulatory properties that can delay optimal protective host immune responses. In this context, this review focuses on selected macrophage immunoregulatory pattern recognition receptors (PRRs), cytokines, negative regulators of inflammation, lipid mediators and microRNAs (miRNAs).

Genetic variation in C-type lectins influences infectious disease susceptibility but remains poorly understood. We used allelic mRNA expression imbalance (AEI) technology for surfactant protein (SP)-A1, SP-A2, SP-D, dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN), macrophage mannose receptor (MRC1) and Dectin-1, expressed in human macrophages and/or lung tissues. Frequent AEI, an indicator of regulatory polymorphisms, was observed in SP-A2, SP-D and DC-SIGN. AEI was measured for SP-A2 in 38 lung tissues using four marker single-nucleotide polymorphisms (SNPs) and was confirmed by next-generation sequencing of one lung RNA sample. Genomic DNA at the SP-A2 DNA locus was sequenced by Ion Torrent technology in 16 samples. Correlation analysis of genotypes with AEI identified a haplotype block, and, specifically, the intronic SNP rs1650232 (30% minor allele frequency); the only variant consistently associated with an approximately twofold change in mRNA allelic expression. Previously shown to alter a NAGNAG splice acceptor site with likely effects on SP-A2 expression, rs1650232 generates an alternative splice variant with three additional bases at the start of exon 3. Validated as a regulatory variant, rs1650232 is in partial linkage disequilibrium with known SP-A2 marker SNPs previously associated with risk for respiratory diseases including tuberculosis. Applying functional DNA variants in clinical association studies, rather than marker SNPs, will advance our understanding of genetic susceptibility to infectious diseases.

Complement receptor 3 (CR3, CD11b/CD18) is a major macrophage phagocytic receptor. The biochemical pathways through which CR3 regulates immunologic responses have not been fully characterized. Francisella tularensis is a remarkably infectious, facultative intracellular pathogen of macrophages that causes tularemia. Early evasion of the host immune response contributes to the virulence of F. tularensis and CR3 is an important receptor for its phagocytosis. Here we confirm that efficient attachment and uptake of the highly virulent Type A F. tularensis spp. tularensis strain Schu S4 by human monocyte-derived macrophages (hMDMs) requires complement C3 opsonization and CR3. However, despite a>40-fold increase in uptake following C3 opsonization, Schu S4 induces limited pro-inflammatory cytokine production compared with non-opsonized Schu S4 and the low virulent F. novicida. This suggests that engagement of CR3 by opsonized Schu S4 contributes specifically to the immune suppression during and shortly following phagocytosis which we demonstrate by CD11b siRNA knockdown in hMDMs. This immune suppression is concomitant with early inhibition of ERK1/2, p38 MAPK and NF-κB activation. Furthermore, TLR2 siRNA knockdown shows that pro-inflammatory cytokine production and MAPK activation in response to non-opsonized Schu S4 depends on TLR2 signaling providing evidence that CR3-TLR2 crosstalk mediates immune suppression for opsonized Schu S4. Deletion of the CD11b cytoplasmic tail reverses the CR3-mediated decrease in ERK and p38 activation during opsonized Schu-S4 infection. The CR3-mediated signaling pathway involved in this immune suppression includes Lyn kinase and Akt activation, and increased MKP-1, which limits TLR2-mediated pro-inflammatory responses. These data indicate that while the highly virulent F. tularensis uses CR3 for efficient uptake, optimal engagement of this receptor down-regulates TLR2-dependent pro-inflammatory responses by inhibiting MAPK activation through outside-in signaling. CR3-linked immune suppression is an important mechanism involved in the pathogenesis of F. tularensis infection.

Acquiring iron (Fe) is critical to the metabolism and growth of Mycobacterium tuberculosis. Disruption of Fe metabolism is a potential approach for novel antituberculous therapy. Gallium (Ga) has many similarities to Fe. Biological systems are often unable to distinguish Ga(3+) from Fe(3+). Unlike Fe(3+), Ga(3+) cannot be physiologically reduced to Ga(2+). Thus, substituting Ga for Fe in the active site of enzymes may render them nonfunctional. We previously showed that Ga inhibits growth of M. tuberculosis in broth and within cultured human macrophages. We now report that Ga(NO3)3 shows efficacy in murine tuberculosis models. BALB/c SCID mice were infected intratracheally with M. tuberculosis, following which they received daily intraperitoneal saline, Ga(NO3)3, or NaNO3. All mice receiving saline or NaNO3 died. All Ga(NO3)3-treated mice survived. M. tuberculosis CFU in the lungs, liver, and spleen of the NaNO3-treated or saline-treated mice were significantly higher than those in Ga-treated mice. When BALB/c mice were substituted for BALB/c SCID mice as a chronic (nonlethal) infection model, Ga(NO3)3 treatment significantly decreased lung CFU. To assess the mechanism(s) whereby Ga inhibits bacterial growth, the effect of Ga on M. tuberculosis ribonucleotide reductase (RR) (a key enzyme in DNA replication) and aconitase activities was assessed. Ga decreased M. tuberculosis RR activity by 50 to 60%, but no additional decrease in RR activity was seen at Ga concentrations that completely inhibited mycobacterial growth. Ga decreased aconitase activity by 90%. Ga(NO3)3 shows efficacy in murine M. tuberculosis infection and leads to a decrease in activity of Fe-dependent enzymes. Additional work is warranted to further define Ga's mechanism of action and to optimize delivery forms for possible therapeutic uses in humans.

BACKGROUND: New evidence links nicotine to the regulation of T cell-mediated inflammation via a 7 nicotinic cholinergic receptor activation, and chronic nicotine exposure (smoking) reduces the incidence of granulomatous diseases. We sought to determine whether nicotine treatment was well tolerated while effectively normalizing immune responses in patients with active pulmonary sarcoidosis.

METHODS: Consenting adults with symptomatic sarcoidosis (n 5 13) were randomly assigned to receive 12 weeks of nicotine treatment plus conventional therapy or conventional therapy alone. Obtained blood cells were evaluated for their responsiveness to selected Toll-like receptor (TLR) and nucleotide oligomerization domain-like receptor ligands and T cell surface marker expression before and after nicotine treatment. Asymptomatic patients (n 5 6) and disease-free subjects (n 5 6) served as comparative control subjects. Adverse events were monitored for the duration of the study.

RESULTS: Compared with the asymptomatic group, symptomatic patients had impaired peripheral responses to TLR2, TLR4, and TLR9 ligands (anergy) and reduced peripheral populations of CD4 1 FoxP3 1 regulatory T cells (Tregs). Nicotine treatment was associated with restoration of TLR2 and TLR9 responsiveness, and expansion of Tregs, including the CD4 1 CD25 2 FoxP3 1 phenotype. There were no serious adverse events or signs of nicotine dependency.

CONCLUSIONS: Nicotine treatment in active pulmonary sarcoidosis was well tolerated and restored peripheral immune responsiveness to TLR2 and TLR9 agonists and expansion of FoxP3 1 Tregs, including a specific “preactivated” (CD25 2 ) phenotype. The immune phenotype of patients with symptomatic sarcoidosis treated with nicotine closely resembled that of asymptomatic patients, supporting the notion that nicotine treatment may be beneficial in this patient population.

Granulomas are the hallmark of Mycobacterium tuberculosis (M.tb) infection and thus sit at the center of tuberculosis (TB) immunopathogenesis. TB can result from either early progression of a primary granuloma during the infection process or reactivation of an established granuloma in a latently infected person. Granulomas are compact, organized aggregates of immune cells consisting of blood-derived infected and uninfected macrophages, foamy macrophages, epithelioid cells (uniquely differentiated macrophages), and multinucleated giant cells (Langerhans cells) surrounded by a ring of lymphocytes. The granuloma's main function is to localize and contain M.tb while concentrating the immune response to a limited area. However, complete eradication does not occur since M.tb has its own strategies to persist within the granuloma and to reactivate and escape under certain conditions. Thus M.tb-containing granulomas represent a unique battlefield for dictating both the host immune and bacterial response. The architecture, composition, function, and maintenance of granulomas are key aspects to study since they are expected to have a profound influence on M.tb physiology in this niche. Granulomas are not only present in mycobacterial infections; they can be found in many other infectious and non-infectious diseases and play a crucial role in immunity and disease. Here we review the models currently available to study the granulomatous response to M.tb.

One-third of the world's population is infected with Mycobacterium tuberculosis (M.tb), which causes tuberculosis. Mycobacterium tuberculosis cell envelope components such as glycolipids, lipoglycans and polysaccharides play important roles in bacteria-host cell interactions that dictate the host immune response. However, little is known about the changes in the amounts and types of these cell envelope components as the bacillus divides during in vitro culture. To shed light on these phenomena, we examined growth-dependent changes over time in major cell envelope components of virulent M.tb by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, thin-layer chromatography, mass spectrometry, immunoblotting and flow cytometry. Our studies provide evidence that major mannosylated glycoconjugates on the M.tb cell envelope change as M.tb grows in vitro on the widely used Middlebrook 7H11 agar. In particular, our compositional analyses show that from Day 9 to 28 the amounts of mannose-containing molecules, such as mannose-capped lipoarabinomannan, lipomannan and phosphatidyl-myo-inositol mannosides, change continuously in both the cell envelope and outer cell surface. Along with these changes, mannan levels on the outer cell surface also increase significantly over time. The implications of these differences in terms of how M.tb is grown for studies performed in vitro and in vivo for assessing M.tb-host recognition and establishment of infection are discussed.

The re-emerging importance of type 2 diabetes mellitus (DM) to tuberculosis (TB) control is of growing concern, but the basis for this relationship is poorly understood. Given the importance of mononuclear phagocytes for TB control and the reported alterations in monocytes of DM patients, we evaluated whether the initial interaction between both was affected in diabetics. Mycobacterium tuberculosis-naïve individuals with and without DM were group matched by age and gender and the efficiency of M. tuberculosis association (attachment and ingestion) with their monocytes was assessed in the presence of autologous serum. The association of M. tuberculosis with monocytes was significantly lower in diabetics (19.2 ± 6.1) than non-diabetics (27.5 ± 7.9; p = 0.02). Multivariate analysis controlling for host socio demographics, DM characteristics and serum lipids indicated that male gender (p = 0.04) and poorly-controlled DM (high HbA1c and hyperglycemia; p = 0.01) were significantly associated with the lower interaction of M. tuberculosis with monocytes. Serum heat-inactivation reduced the association of M. tuberculosis to similar levels in both study groups (p = 0.69) suggesting alterations in the complement pathway of DM patients. These findings suggest an altered route of entry of the pathogen in DM patients that may influence the downstream activation of signaling pathways in the monocyte and the survival of mycobacteria.